The crux of gene therapy for cystic fibrosis is to transfer to the relevant airway epithelial cells an expression cassette containing the coding sequence of the normal human CFTR gene in a fashion that will re- establish normal CFTR function in the airway epithelium. Independent of the vector system used, there are three important elements to be transferred: the CFTR coding sequences, the 3' mRNA maturation signal, and the 5' promoter that supports the expression of these sequences. All available evidence suggests that the 5' promoter element appropriate to correct the airway epithelial CFTR deficiency state in an effective and safe fashion. In this context, the overall goal of this project is to identify promoters that will maximize the efficacy and safety of transfer of the human CFTR cDNA to the airway epithelium, in order to normalize CFTR function in the airways of individuals with cystic fibrosis on a chronic basis. The E1- replication deficient recombinant adenovirus with the promoters and relevant gene inserted in the E1 position will be used as the "model" vector in all studies. Among the classes of promoters and/or promoter elements to be characterized include: other constitutive viral promoters; 5 flanking regions of the normal CFTR gene; epithelial tissue specific promoters; and promotor elements that can be regulated by internal or exogenous stimuli relevant to enhancing efficacy and safety. Replication deficient adenovirus vectors will be constructed using promoters in each class driving the expression of a reporter gene or the normal human CFTR cDNA. These constructs will be used to define the relative amount of expression supported by promoters in each class in vitro and in vivo and characterize the ability of promoters in each class to support the expression of the normal human CFTR cDNA in a fashion sufficient to correct the deficiency of cAMP-inducible Cl- secretion in the airway epithelial cells from individuals with cystic fibrosis. These various classes of promoters in the adenovirus constructs will also be evaluated for their function in airway epithelium in the context of the inflammatory milieu of the airways in cystic fibrosis. Finally, based on the information derived, chimeric promoters will be constructed with features that will optimize the efficacy and safety of in vivo gene therapy for the respiratory manifestations of cystic fibrosis.